Dual Role of the Active Site Residues of <i>Thermus thermophilus</i> 3-Isopropylmalate Dehydrogenase: Chemical Catalysis and Domain Closure

Gráczer, Éva; Szimler, Tamás; Garamszegi, Anita; Konarev, Petr V.; Lábas, Anikó; Oláh, Julianna; Palló, Anna; Svergun, Dmitri I.; Merli, Angelo; Závodszky, Péter; Weiss, M.S.; Vas, Mária

doi:10.1021/acs.biochem.5b00839

Cited by 2 publications

(3 citation statements)

References 61 publications

(151 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…7; Table 3). This suggestion is also consistent with pH effects associated with mutation of the catalytic lysine in the T. thermophilus IPMDH and with quantum mechanical/molecular mechanics simulations suggesting a similar mechanism involving an active site water molecule (28). Moreover, the loss of activity observed in the AtIPMDH2 N234A mutant may result from altered positioning of the water molecule.…”

Section: Discussionsupporting

confidence: 85%

“…5) indicates that the C4-position of the cofactor is 3.0 Å from the substrate. Recent pK a calculations and quantum mechanical/molecular mechanics simulations suggest that the protonation state of the lysine in the T. thermophilus IPMDH active site depends on local environment and favors an unprotonated amine in the resting state (28). The key role for Lys-232 in the reaction mechanism is supported by enzyme assays indicating a loss of activity for the K232M mutant, the x-ray crystal structure of the K232M mutant that shows structural changes in the active site largely limited to the introduced residue (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…It is not clear whether this occurs in solution following release of products or within the enzyme active site. In the bacterial IPMDH and isocitrate dehydrogenase, unusual pH profiles for enzymes with point mutations in the tyrosine have been interpreted as evidence for this residue serving as a general acid in the protonation of the enolate intermediate after decarboxylation (25,28).…”

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

Structure and Mechanism of Isopropylmalate Dehydrogenase from Arabidopsis thaliana

Lee

Nwumeh²,

Jez

2016

Journal of Biological Chemistry

View full text Add to dashboard Cite

Isopropylmalate dehydrogenase (IPMDH) and 3-(2-methylthio)ethylmalate dehydrogenase catalyze the oxidative decarboxylation of different ␤-hydroxyacids in the leucine-and methionine-derived glucosinolate biosynthesis pathways, respectively, in plants. Evolution The evolution of specialized metabolic pathways from primary metabolism provides plants with the ability to generate molecules that contribute to their survival (1). The classic cycle of gene duplication and divergence of sequence that leads to new substrate specificities is at the core of how plants diversify metabolism for new purposes. One example of this process is the evolution of enzymes from leucine biosynthesis into variants for the production of sulfur-containing glucosinolates in plants of the order Brassicales (2-4). In the biosynthesis of methionine-derived glucosinolates, the sequential addition of methylene groups that leads to elongated aliphatic glucosinolates mimics the reactions in leucine biosynthesis (2).In the leucine biosynthesis pathway of plants and microbes, the NAD ϩ -dependent enzyme isopropylmalate dehydrogenase (IPMDH) 3 catalyzes the oxidation and decarboxylation of 3-isopropyl-L-malate (IPM) to 4-methyl-2-oxovalerate ( Fig. 1) (2). Subsequent transamination of 4-methyl-2-oxovalerate produces leucine. In the synthesis of aliphatic glucosinolate biosynthesis, the corresponding 3-malate derivative (i.e. 3-(2Ј-methylthio)ethylmalate) is produced from methionine. Branched-chain aminotransferases catalyze the deamination of methionine to 4-methythio-2-oxobutanoic acid (5, 6). Subsequent steps performed by methylthioalkylmalate synthase and an isopropylmalate isomerase homolog generate 3-(2Ј-methylthio)ethylmalate) (7, 8), which undergoes oxidation and decarboxylation to yield 5-methylthiol-2-oxopentoate ( Fig. 1) (9, 10). This product can then be transaminated for further elongation of the aliphatic moiety to yield C4 to C8 aliphatic glucosinolates (2).In plants, complementation of yeast with a Leu2 mutation by genes from canola, potato, and Arabidopsis thaliana identified IPMDH in the leucine biosynthesis pathway (11-13). Later studies of the three IPMDH isoforms in Arabidopsis (AtIPMDH1-3) revealed differences in the biochemical properties and metabolic contributions of each protein (9, 10). Steady-state kinetic analysis of AtIPMDH1-3 showed that each enzyme catalyzed the conversion of 3-isopropylmalate to 4-methyl-2-oxovalerate; however, the catalytic efficiency of AtIPMDH1 was up to 40-fold lower than the two other isoforms (9, 10). Analysis of Arabidopsis T-DNA insertion mutants that disrupted AtIPMDH1 showed decreased levels of C4 -C8 aliphatic glucosinolates and leucine. The loss of glucosinolate synthesis could be complemented by expression of AtIPMDH1 but not by expression of either AtIPMDH2 or AtIPMDH3 (10). T-DNA mutants of AtIPMDH2 and AtIPMDH3 reduced leucine levels but did not significantly alter glucosinolate production in Arabidopsis (14). Moreover, the Arabidopsis AtIPMDH2/AtIPMDH3 double mutant had defects in...

show abstract

Section: Discussionsupporting

confidence: 85%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation